Plant viruses induce plant volatiles that are detected by aphid parasitoids

Aphis gossypii (Sternorrhyncha: Aphididae) aphids are vectors of important plant viruses among which cucumber mosaic virus (CMV) and potato virus Y (PVY). Virus-infected plants attract aphid vectors and affect their behavior and growth performance either positively or negatively depending on mode of transmission. Viruses cause changes in the composition and the amount of volatile organic compounds (VOCs) released by the plant that attract aphids. The aphid parasitoid Aphidius colemani (Hymenoptera: Aphelinidae) has been shown to have higher parasitism and survival rates on aphids fed on virus-infected than aphids fed on non-infected plants. We hypothesized that parasitoids distinguish virus-infected plants and are attracted to them regardless of the presence of their aphid hosts. Herein, we examined the attraction of the A. colemani parasitoid to infected pepper plants with each of CMV or PVY without the presence of aphids. The dynamic headspace technique was used to collect VOCs from non-infected and CMV or PVY-infected pepper plants. Identification was performed with gas chromatography-mass spectrometry (GC–MS). The response of the parasitoids on virus-infected vs non-infected pepper plants was tested by Y-tube olfactometer assays. The results revealed that parasitoids displayed a preference to CMV and PVY infected plants compared to those that were not infected.


Results
Response to olfactometer. To test the attraction of adult parasitoids towards virus-infected pepper plants we performed pair-wise preference tests in which parasitoids were exposed to a combination of either virus infected plants and non-infected plants or virus-infected plants and clean air. There was no significant effect of the species of virus on the response of the parasitoids (GLM; χ 2 = 0.0, df = 1, P = 1). Parasitoids were more attracted to CMV-infected pepper plants versus non-infected pepper plants (GLM; χ 2 = 11.59, df = 1, P = 0.001) as well as to PVY-infected pepper plants versus non-infected pepper plants (GLM; χ 2 = 4.88, df = 1, P = 0.027) (Fig. 1). Parasitoids were also more attracted to CMV-infected pepper plants versus clean air (GLM; χ 2 = 69.66, df = 1, P < 0.001) as well as to PVY-infected pepper plants versus clean air (GLM; χ 2 = 33.79, df = 1, P < 0.001) (Fig. 1). Table S1). The VOCs profiles of CMV and PVY infected plants differ quantitatively and qualitatively to those of non-infected plants ( Fig. 2; Supporting information Table S1, Figs. S1, S2). There is a tendency for lower emission of VOCs in virus infected pepper plants but that was not found to be statistically significant (χ 2 = 5.671, df = 2, p = 0.059). In CMV-infected pepper plants 18 VOCs were identified that were not detected in non-infected and PVY-infected pepper plants. CMV-infected pepper plants emitted hexanal, terpenes (α-pinene, limonene), sesquiterpenes (β-elemene, β-longipinene) and homoterpene (Ε)-4,8,12-trimethyl-1,3,7,11-tridecatetraene [(Ε)-ΤΜΤΤ] which were absent from non-infected and PVY-infected pepper plants. In non-infected pepper plants, 6 VOCs have been identified that were not found in virus-infected pepper plants. The chemometric analysis showed that virus-infected and non-infected pepper plants are separated based on volatiles emitted (Fig. 3a). The first two principal components explained 41.9% and 15.6% of the variance, respectively. According to the loading plot, the first principal component separates CMV paper plants from the two other treatments, while second principal component clearly separates virus infested pepper plants from control ones (Fig. 3b). The model identified 21 compounds with variable importance for the projection (VIP) values > 1 (Table 1). Interestingly, the volatile emissions of 11 compounds out of the total 21 shown in Table S1 were significantly different among treatments. Virus infected pepper plants were mostly correlated with the emissions of ester 1, ester 2, ester 4, heptadecane, 2,6,10-trimethyl-dodecane and unknown 6. VIP compounds that characterize control pepper plants are 2,2,4,6,6-pentamethyl heptane, 2-ethyl-1-hexanol, alkane 3, undecane, alkane 10, dodecane and 2,4-dimethyl heptane. Further pairwise PLS-DA models between the blends emitted by plants infested by CMV and the control plants and PVY versus control plants were carried out. PLS-DA analysis yielded a separation between CMV-infested and control plants (Fig. 4). In total, 24 compounds contributed most to the separation ( www.nature.com/scientificreports/ and sesquiterpene β-elemene, that were detected only in CMV-infected pepper plants, were positively correlated to CMV-infected pepper plants. Figure 5 shows the separation between PVY-infected plants and control pepper plants. In this case, 22 compounds had a VIP value higher than 1 (Table 3). Ester 4, 2,6,10-trimethyl-dodecane, heptadecane, alkane 4, ester 2 and ester 1 were influenced by PVY-infection. These compounds found in higher levels also in the headspace pf PVY-infected plants (Table S1).

Discussion
Plants respond to insect herbivory by enhancing their defense mechanisms either directly or indirectly 42     www.nature.com/scientificreports/ this interaction by increasing their foraging efficiency and parasitization success 46 , although the opposite is not excluded 47 . Vector-transmitted pathogens have been proven to alter the volatile profile of host plants to attract vectors and consequently enhancing pathogen dispersal and proliferation 11,13 . However, this is a species specific interaction as in other plant virus combinations no attraction of aphids to virus infected plants was observed 48 . Previously, attraction of parasitoids to virus infected plants has been shown for the whitefly-vectored tomato yellow leaf curl virus (TYLCV) 49 . Attraction of parasitoids to plant pathogen induced volatiles has been shown also for the psyllid parasitoid Tamarixia radiata, that it was attracted to odors released by citrus trees infected by the bacterium Candidatus Liberibacter asiaticus (Las) which causes the citrus greening huanglongbing 30 . In the above studies parasitoid attraction to pathogen infected plants was achieved even without the presence of their herbivore hosts. In the pioneer study of Mauck et al. 34 , A. colemani parasitoids didn't show any preference for the odors of CMV-infected plants or non-infected plants, but another plant species has been used, Cucurbita pepo, and plants were simultaneously infested by M. persicae aphids 34 . The presence of aphids might have obscured the effect of the virus as aphids themselves cause HIPVs that attract parasitoids 36,37 . Similarly, T. radiata parasitoids didn't show any preference for bacterial infected plants when their psyllid hosts were present 30 . In our assays, A. colemani parasitoids were able to discriminate non-infected and virus-infected plants and were attracted to the odors of both CMV and PVY infected plants. In all cases plants were free from their host aphids. To our  www.nature.com/scientificreports/ knowledge, this is the first study that demonstrates a parasitoid species attraction towards virus infected plants without the presence of aphids. Presence of aphids might have confounded the attraction of parasitoids to virus infected plants due to aphids own emitted VOCs 50 or due to aphids induced HIPVS 35,36 . We cannot exclude an increased parasitoids' attraction towards virus-infected plants if they bear the aphid hosts/vectors but nevertheless, our findings are relevant because it is likely that both viruses exist in nature without the presence of aphids as except of aphid transmission, they are either seed or mechanically transmitted 51,52 . The biological or evolutionary drives behind the observed behavior are still obscure as it is difficult to identify a clear advantage exclusively for each of the four parties involved. Thus, parasitoids may benefit by identifying virus infected plants through earlier location of a habitat that is more likely to harbor host plants with aphids and thus resulting in more efficient foraging. Plants may also benefit by attracting natural enemies as they will decrease herbivore abundance in the plant community and consequently virus spread and prevalence. Early arrival of parasitoids as a response to virus-induced volatiles would favor biological control of aphid pests. Successful early biological control of aphids has been associated with reduction of aphid-vectored plant virus as early presence of natural enemies deter establishment of aphid 53 55,58 . Plant viruses as well as insect herbivores elicit plant defence signal-transduction in the jasmonate (JA), salicylic acid (SA) and ethylene (ET) pathways 44,59 . Infection of plants by viruses interferes with the physiological SA and JA defense signaling by creating more favourable conditions for the aphid vectors as well their attraction to the infected plants 6,7 . In our study, CMV infection induced the emission of terpenoids and in some cases the reduction in emission rates of monoterpenoids (camphor, isoborneol). Aphis gossypii infestation induced emission of terpenoids among other VOCs in cotton and cucumber plants 60,61 . Although CMV infected C. pepo plants were found to be nutritionally inferior for A. gossypii, aphids that are attracted to virus-infected plants, might disperse rapidly facilitating the spread of the non-persistent virus 13 . We don't know if CMV or PVY infected pepper plants, are inferior for A. gossypii too. In case they are and aphids are dispersing from the plants, parasitoids will be attracted to plants where it is less likely to find aphid hosts to parasitize, leaving a portion of the vector population free of parasitism resulting in further spread of the virus in the plant community 30 . However, this remains to be confirmed by further experimental tests.
Our results contribute to the understanding of the complex interaction of plants, pathogens insect vectors and their natural enemies. However, in natural environment insects encounter a much more complex array of www.nature.com/scientificreports/ volatiles that they need to utilize for making behavioral decisions. Therefore, further investigation by combining laboratory and semi-field/field studies is needed for understanding the plant-insect interactions and elucidating the role of the virus induced VOCs in the behavior of parasitoids. The identification of potential compounds that act either as attractants or repellents either for the parasitoids or the aphid vectors will facilitate proper pathogen management.

Olfactometer behavioral experiments. Attraction of adult female parasitoids to virus infected pepper
plants was assessed by a Y-tube olfactometer. A combination of a pepper plant infected either by CMV or PVY versus clean air or non-infected (control) pepper plant was offered to the adult parasitoid. The responses were assessed in a glass Y-tube olfactometer with 1 cm internal diameter, 10 cm main arm length and side arms 8 cm long. Plants were introduced into a 10 L glass jar connected with Teflon tubing in each arm. For clean air, an empty jar was used. Air was pumped (Dymax 5, Charles Austen Pumps Ltd, UK) through an active charcoal filter and re-humidified by passing through a bottle with tap water before directed into each jar connected to the one of the two arms of the olfactometer. The olfactometer was lined underneath with filter paper and evenly lightened for uniform lighting. Air flow rate was approximately 60 mL min −1 . For each bioassay, a single female Table 2. Values of variable importance to the projection (VIP) of volatiles for PLS-DA CMV-CON . www.nature.com/scientificreports/ A. colemani was introduced into the central arm of Y-tube and left for 5 min to make a choice. A choice was recorded when a female was crossing 2 cm within the side arm and stayed for 15 s. A wasp which did not make a choice within 5 min was recorded as a 'no response' . Tests were conducted from 10:00 to 14:00 h. In all bioassays, after three runs the test stimulus positions were reversed to avoid any directional bias. After three replicates, the olfactometer was thoroughly washed with soap and water and rinsed with acetone before oven-dried at 120 °C. For each odour combination a single plant was used for each experimental day. Six wasps were used per day and odour combination to form a replicate. At least 22 replicates were performed for each combination.

Collection of volatiles.
Collection of plant volatiles was performed in laboratory. Potted pepper plants 3 weeks old (2 weeks post inoculation for infected plants) were transferred from the greenhouse nursery to the laboratory. The pot of each plant was hermetically covered with aluminum foil to prevent interaction with VOCs from soil and roots. Subsequently, each plant was left for 30 min for acclimatization before being placed in a glass container (10 L). Plants with any sign of mechanical damage were discarded. VOCs collection was performed by dynamic headspace sampling 64 . Ambient air was purified through an activated charcoal filter Identification. Identification of volatiles from headspace extracts was performed using gas chromatography-mass spectrometry (GC-MS). One microliter of the extract was used for the analysis. It was injected in a Varian CP-3800 GC, with a 1079 injector coupled with a 1200L quadrupole mass spectrometer. Separation of the analytes was performed with a TG-5MS capillary column (5% diphenyl/95% dimethyl polysiloxane) with dimensions 30 m length, 0.25 mm i.d., 0.25 μm film thickness (Thermo Scientific, Waltham, USA). Spitless mode was set for 1 min. The flow rate of the carrier gas helium was 1 mL min −1 . The oven temperature was maintained at 50 °C for 5 min, increased with a rate of 3 °C min −1 to 170 °C and with a rate at 20 °C min −1 to the final temperature of 250 °C. Mass spectrometer was operated in Electron ionization mode (EI) with ion energy of − 70 eV, filament current 50 μA and source temperature 200 °C. Data acquisition was performed in full scan (MS) with scanning range 40-300 amu. Tentative identification was achieved by comparing their elution order, the mass spectra with those from mass spectra libraries (Adams 2007, NIST 2005, Wiley 275) and literature data 65,66 . We also used retention indices (RI) of a series of n-alkane (C 8 -C 20 ), using the formula: 100n + 100 [(R t (X) − R t (N))/ (R t (N + 1) − R t (N))], which is based on retention times of linear alkane standards; n = number of carbon atoms of the alkane N; R t (X) = retention time of target compound; R t (N) = retention time of N alkane which elutes before X; R t (N + 1) = retention time of alkane eluting after X. Wherever possible, retention time and mass spectra were compared with commercial standards. The total ion chromatogram was processed by Varian MS Workstation software (version 6.9) based on the retention time and mass spectrum.

Statistical analysis.
To investigate whether parasitoid preference differed between the two combinations of virus-infected plants, data were analysed using logistic regression [i.e. a generalized linear model (GLM)] with a binomial distribution and a logit link function] with virus species as fixed factor. A quasi-binomial distribution was fitted in the model due to overdispersion. Since one plant was used for each experimental day and six wasps were used per day and odour combination to form a replicate, we used as response variable the number of wasps choosing the virus-infected plants out of the total number of responding wasps 67 . To determine under dualchoice conditions whether there was a significant preference for one of the offered plant treatments, we used (GLM) with a binomial distribution and a logit link function with virus treatment as fixed factor. The number Table 3. Values of variable importance to the projection (VIP) of volatiles for PLS-DA PVY-CON . www.nature.com/scientificreports/ of wasps choosing the virus-infected plants out of the total number of responding wasps was used as response variable. Data were analysed with SPSS. Non-responding individuals were excluded from statistical analyses. Volatile compounds, measured as peak area and normalized with the peak area of internal standard, were log-transformed and processed by projections to latent structures-discriminant analysis (PLS-DA) using SIMCA 16.0 software (Umetrics, Umeå, Sweden). The Pareto scaling method was applied to the dataset before PLS-DA processing. Additionally, compounds with a variable importance for the projection (VIP) value higher than 1 were also generated. VIP values estimate the importance of each variable (compound) in the projection and is often used for variable selection 68 . Non-parametric Kruskal-Wallis (SPSS) was performed to identify differences in the quantities of the total VOCs amount and for each identified compound among different plant treatments.

Data availability
Most of the data generated or analysed during this study are included in this published article (and its Supplementary Information files). All other datasets generated during and/or analysed during the current study are available from the corresponding author on request.